Fatty acid analogues for the treatment of primary and secondary restenosis

ABSTRACT

The present invention relates to novel fatty acid analogues of the general formula (I): CH 3 —[CH 2 ] m —[X i —CH 2 ] n —COOR, wherein n is an integer from 1 to 12, and wherein m is an integer from 0 to 23, and wherein i is an odd number which indicates the position relative to COOR, and wherein X i  independent of each other are selected from the group comprising O, S, SO, SO 2 , Se and CH 2 , and wherein R represents hydrogen or C 1 -C 4  alkyl, with the proviso that at least one of the X i  is not CH 2 , or a salt, prodrug or complex thereof, for the preparation of a pharmaceutical composition for the treatment and/or prevention of primary and/or secondary stenosis. Further the present invention relates to the use of said compounds for the prevention and/or treatment of a disease caused by procedural vascular trauma and/or pathological proliferation of smooth muscle cells, and/or an increased level of plasma homocysteine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §120 to PCT No.PCT/NO99/00149, filed May 8, 1999.

BACKGROUND OF THE INVENTION

The present invention relates to novel fatty acid analogues which can beused for the treatment and/or prevention of primary and secondarystenosis. Further, the present invention relates to the use of saidanalogues for the treatment of diseases caused by procedural vasculartrauma, and more specifically the invention relates to conditionsassociated with smooth muscle cell proliferation.

Many pathological conditions have been found to be associated withsmooth muscle cell proliferation. Such conditions include restenosis,atherosclerosis, coronary heart disease, thrombosis, myocardialinfarction, stroke, smooth muscle neoplasms such as leiomyoma andleiomyosarcoma of the bowel and uterus and uterine fibroid or fibroma.

Over half a million interventional intravascular procedures areperformed each year. While such invasive procedures continue to improveover time, as many as 30-50% of the procedures performed each year failas a result of restenosis, i.e. the formation of secondary stenosis. Thereduction of restenosis is, therefore, often cited as the most criticalfactor in increasing the success realised in the treatment ofcardiovascular disease through the use of interventional intravascularprocedures, such as angioplasty, atherectomy, and procedures utilisingstents and laser technology.

In balloon angioplasty, e.g. Percutaneous Transluminal CoronaryAngioplasty (PTCA), a small incision is made to an artery in thepatient's leg or arm and a long hollow tube, called a guide catheter, isinserted into the artery. A thick guide wire and deflated ballooncatheter are then inserted into the guide catheter and are carefullyadvanced through the patient's blood vessels using x-ray visualisation.The deflated balloon is advanced until it reaches the site of theluminal narrowing, at which point the physician inflates the balloon oneor more times to a pressure of about 4-6 atm for about 60 sec. Wheninflated, the balloon cracks and fractures the plaque and stretches themuscle fibre in the artery wall beyond its ability to recoil completely.Although no plaque is removed in this procedure, the fracturing of theplaque and the stretching of the arterial wall increase the vessellumen, thereby allowing for increased blood flow.

The restenosis that accompanies such procedures is characterised byplatelet aggregation and adhesion, smooth muscle cell proliferation,narrowing of the vessel lumen, restricted vasodilatation, and anincrease in blood pressure. Smooth muscle cells in the intimal layer ofthe artery have been reported to enter the growth cycle within about 2-3days of these procedures and to proliferate for several days thereafter(intimal hyperplasia).

Compounds that reportedly suppress smooth muscle proliferation in vitromay have undesirable pharmacological side effects when used in vivo.Heparin is an example of one such compound, which reportedly inhibitssmooth muscle cell proliferation in vitro but when used in vivo has thepotential adverse side effect of inhibiting coagulation.

As is apparent from the foregoing, many problems remain to be solved inthe use of inhibitory drugs to effectively treat smooth muscle cellmobilisation and proliferation. It would be highly advantageous todevelop new compositions or methods for inhibiting stenosis, restenosisor related disorders due to proliferation and mobilisation of vascularsmooth muscle cells following, for example, traumatic injury to vesselsrendered during vascular surgery.

Treatment with modified fatty acids represent a new way to treat thesediseases.

EP 345.038 and PCT/NO95/00195 describes the use of non-β-oxidizablefatty acid analogues for the treatment of hyperlipidemic conditions.

It has now been found that these fatty acid analogues have broader areaof applications.

Further, we have now synthesised and characterised novel fatty acidanalogues.

In feeding experiments with the fatty acid analogues of the presentinvention, the results show that these compounds lower the adiposetissue mass and body weight, and are thus potent drugs for the treatmentof obesity and overweight. These results are described and claimed inthe Applicants co-pending application PCT/NO99/00135.

We have also shown that the fatty acid analogues are potent antidiabeticcompounds, with a profound effect on the levels of glucose and insulin.These results are described and claimed in the Applicants co-pendingapplication PCT/NO99/0136.

We have shown that the compounds of the present invention inhibit theformation of secondary stenosis, and the present application thusrelates to the use of these compounds for the prevention and/ortreatment of restenosis. Further, we have shown that the compoundsinhibit the proliferation and mobilisation of smooth muscle cells, andlower the concentration of plasma homocysteine. It is thus anticipatedthat the compounds also will have a preventive and/or therapeutic effecton primary stenosis. Further, it is anticipated that the presentcompounds will be useful for the treatment and/or prevention ofatherosclerosis, coronary heart disease, thrombosis, myocardialinfarction, stroke and smooth muscle cell neoplasms, and also diseasescaused by procedural vascular trauma.

The novel compounds of the present invention are characterised by minormodifications of the natural fatty acids. Sulphur, selenium or oxygenare preferably substituted for one or more of the carbons in the fattyacid backbone. The compounds defined by the formula I have propertieswhich give them a unique combination of biological effects.

Tetradecylthioacetic acid (TTA) and tetradecylselenioacetic acid (TSA)are most thoroughly studied, and we have shown several beneficialeffects in various model animal systems.

The studies have shown that TTA has properties very similar to naturalfatty acids, the main difference being that TTA is not oxidised by themitochondrial β-oxidation system. However, the presence of compounds ofthe present invention have been shown to increase the β-oxidation ofother (non-substituted) fatty acids.

Administration of TTA to rats for 12 weeks nearly doubled the hepaticand plasma content of monounsaturated fatty acids (mainly oleic acid),while polyunsaturated fatty acids (mainly linoleic acid and DHA)decreased. Thus the compound of the present invention modifies thecomposition of the lipids in various tissues.

Feeding moderate doses of TTA to animals like rats, mice, rabbits anddogs decreased both plasma cholesterol and triacylglycerol levels withindays of treatment. We have also shown the same effect for TSA, andcompounds of the present invention with Sulphur substituted in positions5 or 7 have been shown to increase the β-oxidation, and it is thusanticipated that also these fatty acid analogues will lower the plasmalevels of triacylglycerols and cholesterol. TTA and TSA are far morepotent in this respect than polyunsaturated fatty acids like EPA.

The experimental data of the present invention have unexpectedlyrevealed that the formation of secondary stenosis (restenosis) afterangioplasty is markedly reduced or inhibited in various model animalsgiven the compounds of formula I either orally or locally. This isclearly demonstrated in the experimental section, examples 3 and 4,which demonstrates that the artery diameter, several weeks after theangioplasty procedure, is maintained for animals given TTA, while thediameter is markedly reduced for control animals. These in vivo resultsclearly demonstrate the potential of these compounds for the preventionof the formation of secondary stenosis.

The action mechanisms for the formation of restenosis after PTCA are notcompletely understood, but it have been shown that restenotic lesionshas an overgrowth of smooth muscle cells in the intimal layers of thevessel.

We have shown that the compounds of the present invention reduce thegrowth and mobilisation of smooth muscle cells. Increased smooth musclecell proliferation has also been associated with atherosclerosis,coronary heart disease, thrombosis, myocardial infarction and stroke.

Normal blood vessels are lined with a layer of endothelial cells. Theendothelium releases local factors such as nitric oxide, prostaglandinI₂ and prostacyclin into the vessel wall (intramural release) and intothe blood stream (intraluminal release). These factors maintain vasculartone (vessel relaxation), inhibit clot formation on the vessel innersurface (platelet adhesion and aggregation), inhibit monocyte adherenceand chemotaxis, and inhibit smooth muscle cell migration andproliferation. As a result of this process, vasodilation andthrombolysis occurs, and blood flow is maintained. If the endothelium isdysfunctional or damaged, however, nitric oxide and prostacyclin releaseis impaired. Platelet aggregation and adhesion can occur unopposed, withplatelet-derived products acting directly on the smooth muscle cells tocause vasoconstriction. The net result is a blood vessel which is highlysusceptible to thrombosis and vasospasm.

Atherosclerosis can form within a blood vessel over a period of yearsfrom a variety of causes. The resulting lesion, or plaque, mayprogressively occlude the vessel and impede blood flow to vital organs.

The described vasoconstrictive physiologic mechanisms occur both inperipheral and in coronary arteries, but the consequences of theprocesses are more life threatening in the coronary arteries. Coronaryarteries, the arteries of the heart, perfuse the cardiac muscle withoxygenated arterial blood. They provide essential nutrients and allowfor metabolic waste and gas exchange. These arteries are subject tounremitting service demands for continuous blood flow throughout thelife of the patient. A severe proximal coronary stenosis withendothelial injury induces cyclic coronary flow reductions (“CFR's”).These are periodic or spasmodic progressive reductions in blood flow inthe injured artery. Episodes of CFR's are correlated to clinical acuteischemic heart disease syndromes, which comprise unstable angina, acutemyocardial infarction and sudden death. The common pathophysiologic linkis endothelial injury with vasospasm and/or thrombus formation.

It is thus anticipated and claimed that the compounds of the presentinvention by its action on the smooth muscle cells will have afavourable effect on the group of diseases mentioned above.

As indicated, the present compounds also exhibit a lipid loweringeffect, and the administration of a compound of the present invention isthus the compound of choice for the treatment or prevention of arteryrelated diseases. The low toxicity of these compounds makes them verysuitable as prophylactically agents given to mammals in need thereof,e.g. to prevent the formation of primary stenosis.

We have also demonstrated that the compounds of the present inventiondecrease the plasma concentration of homocysteine. Elevated levels ofhomocysteine are considered as a risk factor for, and are correlatedwith the development of atherosclerosis. Thus, based on thishomocysteine lowering effect of TTA it is anticipated that the presentcompounds will have a inhibiting effect on the formation of primarystenosis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses that modified fatty acid analogues atnon-cytotoxic concentrations can be used for the treatment and/orprevention of stenosis and restenosis.

We have also shown that the compounds of the present invention reducesthe proliferation and mobilisation of smooth muscle cells, and it isknown that proliferation of smooth muscle cells is a pathological factorin diseases such as atherosclerosis, coronary heart disease, thrombosis,myocardial infarction, stroke and smooth muscle cell neoplasms, and thetreatment and/or prevention of such diseases by the use of the compoundsof formula (I) are also part of the present invention.

The present invention relates to the use of fatty acid analogues of thegeneral formula (I):

CH₃—[CH₂]_(m)—[X_(i)—CH₂]_(n)—COOR

wherein n is an integer from 1 to 12, and

wherein m is an integer from 0 to 23, and

wherein i is an odd number which indicates the position relative toCOOR, and

wherein X_(i) independent of each other are selected from the groupcomprising O, S, SO, SO₂, Se and CH₂, and

wherein R represents hydrogen or C₁-C₄ alkyl,

with the proviso that at least one of the X_(i) is not CH₂,

or a salt, prodrug and complex thereof, for the preparation of apharmaceutical composition for the treatment and/or prevention ofprimary and/or secondary stenosis.

In particular, the invention relates to the use of a compound of thegeneral formula I, for the treatment and/or prevention of a diseasecaused by procedural vascular trauma and/or pathological proliferationof smooth muscle cells and/or an increased level of plasma homocysteine.

Preferred embodiments of the invention relate to the use of a compoundof the general formula I, wherein the diseases are selected from thegroup comprising atherosclerosis, coronary heart disease, thrombosis,myocardial infarction, stroke, vascular dementia and smooth muscle cellneoplasms.

One embodiment of the invention is the use of a compound of formula Iwherein m≧13.

A presently preferred embodiment of the invention comprises the formulaI, wherein X_(i=3) is selected from the group consisting of O, S, SO,SO₂ and Se, and wherein X_(i=5-25) is CH₂.

Tetradecylthioacetic acid (TTA) and Tetradecylselenoacetic acid (TSA),i. e. X_(i=3) is Sulphur and Selenium, respectively are presentlypreferred compounds.

A further aspect of the invention relates to a method for theprophylactic or therapeutic treatment of primary or secondary stenosisin a mammal, said method comprising the step of administering to amammal in need thereof an effective amount of fatty acid analogues ofthe general formula (I):

CH₃—[CH₂]_(m)—[X_(i)—CH₂]_(n)—COOR

wherein n is an integer from 1 to 12, and

wherein m is an integer from 0 to 23, and

wherein i is an odd number which indicates the position relative toCOOR, and

wherein X_(i) independent of each other are selected from the groupcomprising O, S, SO, SO₂, Se and CH₂, and

wherein R represents hydrogen or C₁-C₄ alkyl,

with the proviso that at least one of the X_(i) is not CH₂,

or a salt, prodrug or complex thereof.

The treatment involves administering to a patient in need of suchtreatment an effective concentration which is maintained substantiallycontinuously in the blood for the duration of the period of itsadministration.

Further, the invention relates to a pharmaceutical composition for theprevention and/or treatment of a primary and/or secondary stenosis.Preferably, the pharmaceutical composition comprises in admixture withthe fatty acid analogues a pharmaceutically acceptable carrier orexcipient.

The invention also relates to novel fatty acid analogues of the formulaI

CH₃—[CH₂]_(m)—[X_(i)CH₂]_(n)—COOR

wherein n is an integer from 1 to 12, and

wherein m is an integer from 0 to 23, and

wherein i is an odd number which indicates the position relative toCOOR, and

wherein X_(i) independent of each other are selected from the groupcomprising O, S, SO, SO₂, Se and CH₂, and

wherein R represents hydrogen or C₁-C₄ alkyl,

with the proviso that at least one of the X_(i) is not CH₂,

or a salt, prodrug or complex thereof.

FIGURE LEGENDS

FIG. 1 shows an iliac artery of a control animal, 6 weeks after ballooninjury.

FIG. 2 shows an iliac artery from an animal given TTA, six weeks afterballoon injury.

FIG. 3 shows the inhibiting effect of TTA on the growth of smooth musclecells.

DEFINITIONS

Primary Stenosis

The term stenosis refers to a stricture of any canal, and especially anarrowing of one of the cardiac valves. Primary stenosis refers to theformation of stenosis due to a disease condition, e.g. atherosclerosis.

Secondary Stenosis (Restenosis)

The terms <<secondary stenosis>> or <<restenosis>> refers to therecurrence of stenosis after corrective surgery, i.e. narrowing of astructure (usually a coronary artery) following the removal or reductionof a previous narrowing.

Procedural Vascular Trauma

The term procedural vascular trauma refers to conditions caused byvascular surgical procedures such as angioplasty, atheroectomy,placement of stent (e.g. in a vessel), thrombectomy and grafting.

Proliferation

As used herein the term <<proliferation>> means an increase in cellnumber, i.e. by mitosis of the cells.

ADMINISTRATION OF THE COMPOUNDS OF THE PRESENT INVENTION

As a pharmaceutical medicament the compounds of the present inventionmay be administered directly to the mammal by any suitable technique,including parenterally, intranasally, orally, or by absorption throughthe skin. They can be administered locally or systemically. The specificroute of administration of each agent will depend, e.g., on the medicalhistory.

Examples of parenteral administration include subcutaneous,intramuscular, intravenous, intraarterial, and intraperitonealadministration.

As a general proposition, the total pharmaceutically effective amount ofeach of the compounds administered parenterally per dose will preferablybe in the range of 5 mg/kg/day to 1000 mg/kg/day of patient body weight,although, as noted above, this will be subject to a great deal oftherapeutic discretion. For TTA it is expected that a dose of 100-500mg/kg/day is preferable, and for TSA the dosage could range from 10 to100 mg/kg/day.

If given continuously, the compounds of the present invention are eachtypically administered by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The key factor in selecting anappropriate dose is the result obtained.

For parenteral administration, in one embodiment, the compounds of thepresent invention are formulated generally by mixing each at the desireddegree of purity, in a unit dosage injectable form (solution,suspension, or emulsion), with a pharmaceutically acceptable carrier,i.e., one that is non-toxic to recipients at the dosages andconcentrations employed and is compatible with other ingredients of theformulation.

Generally, the formulations are prepared by contacting the compounds ofthe present invention each uniformly and intimately with liquid carriersor finely divided solid carriers or both. Then, if necessary, theproduct is shaped into the desired formulation. Preferably the carrieris a parenteral carrier, more preferably a solution that is isotonicwith the blood of the recipient. Examples of such carrier vehiclesinclude water, saline, Ringer's solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein, as well as liposomes.

The carrier may suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or non-ionicsurfactants such as polysorbates, poloxamers, or PEG.

For oral pharmacological compositions such carrier material as, forexample, water, gelatine, gums, lactose, starches, magnesium-stearate,talc, oils, polyalkene glycol, petroleum jelly and the like may be used.Such pharmaceutical preparation may be in unit dosage form and mayadditionally contain other therapeutically valuable substances orconventional pharmaceutical adjuvants such as preservatives, stabilisingagents, emulsifiers, buffers and the like. The pharmaceuticalpreparations may be in conventional liquid forms such as tablets,capsules, dragees, ampoules and the like, in conventional dosage forms,such as dry ampoules, and as suppositories and the like.

The treatment with the present compounds may occur without, or may beimposed with, a dietary restriction such as a limit in daily food orcalorie intake, as is desired for the individual patient.

A preferred embodiment of the present invention comprises locallyadministration of the compounds. Recently, site-specific drug deliveryto the arterial wall has become a new strategy for the treatment ofvascular diseases, including vessel restenosis following PTCA. Thesedrug delivery systems include: (1) intravascular devices forsite-specific (coronary artery) drug delivery comprising double-ballooncatheters, porous balloon catheters, microporous balloon catheters,channel balloon catheters, balloon over stent catheters, hydrogel coatedballoon catheters, iontophoretic balloon catheters and stent devices;(2) periadventitial and epicardial drug delivery devices, requiringsurgical implantation, which include drug-eluting polymer matrices and aiontophoretic patch device; and (3) intramural injection of drug-elutingmicroparticles.

In addition, the compounds of the present invention are appropriatelyadministered in combination with other treatments for combating orpreventing stenosis.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention.

Experimental Section

Methods and Results

EXAMPLE 1

Preparation and Characterisation of the Compounds

a) Synthesis of the Novel Compounds

Fatty acids with the heteroatom in variable positions were synthesisedaccording to the general description for 3-substituted analogues (seebelow) with the following modification:

Alkyl-Hal was replaced by Alcanoic-Hal and HS—CHCOOR was replaced byalkyl-SH.

The following fatty acid analogues have been prepared and characterised:

Melting- point Compounds Reactants (° C.) Dodecanylthiobutanoic4-bromdbutanoic acid + 54-55 acid dodecanylthiol Decanylthiohexanoic6-brornohexanoic acid + 50-51 acid decanylthiol Octanylthiooctanoic8-bromooctanoic acid + 39-40 acid octanylthiol

Purification of products as described below. Purity >95%. Structure wasverified by mass spectrometry.

b) The Synthesis of the 3-substituted Fatty Acid Analogues

The compounds used according to the present invention wherein thesubstituent X_(i=3) is a sulphur atom or selenium atom may be preparedaccording to the following general procedure:

X is a sulphur atom:

The thio-substituted compound used according to the present inventionmay be prepared by the general procedure indicated below:

The sulphur-compound, namely, tetradecylthioaceticacid (TTA),(CH₃—(CH₂)₁₃—S—CH₂—COOH was prepared as shown in EP-345.038.

X is a selenium atom:

the seleno-substituted compound used according to the present inventionmay be prepared by the following general procedure

This compound was purified by carefully crystallisation from ethanol ormethanol.

The final compound, e.g. when alkyl is tetradecyl,(CH₃—(CH₂)₁₃—Se—CH₂—COOH (tetradecylselinioacetic acid (TSA)) can bepurified by crystallisation from diethyl ether and hexane. This productmay be fully characterised by NMR, IR and molecular weightdetermination.

The methods for the synthesis and isolation of these Sulphur andSelenium compounds, and the compound wherein X of formula I is Oxygen(O), Sulphur-I-oxide (SO) and Sulphurdioxide (S₂) are described inEuropean Patent No. 345.038, and International Patent Application No. WO97/03663.

EXAMPLE 2

Toxicity Study of TTA

A 28 days toxicity study in dogs according to GLP guidelines has beenperformed by Corning Hazleton (Europe), England. Oral administration ofTTA at dose levels up to 500 mg/kg/day was generally well tolerated.Some lipid related parameters were lowered in the animals given highdosages. This is consistent with the pharmacological activity of TTA.

The dose level of 500 mg/kg/day also elicited body weight loss. Therewas no evidence of toxicity at dose levels of 50 or 500 mg/day/kg.

Tests for mutagenic activity have been performed by Covance LaboratoriesLimited, England. It was concluded that TTA and TSA did not inducemutations in strains of Salmonella typhimurium and Escherichia coli.Furthermore, TTA was not mutagenic when tested in mouse lymphoma cellsand L5178Y.

The concentration of the compounds tested in S. typhimurium and E. coliwere 3-1000 mg/plate (TTA) and 2-5000 mg/plate (TSA). In mouse lymphomacells, L5178Y, the concentration was 2,5-50 mg/ml.

TSA and TSA were found not to be mutagenic in these tests. TSA and TTAhave been tested for chromosomal aberrations in cultured Chinese hamsterovary cells and no aberrations were induced by the doses tested (12-140mg/ml).

The compounds of the present invention are therefore potentially usefulas pharmaceutical compounds in this respect.

EXAMPLE 3

Prevention of Restenosis in Rabbits Given TTA Orally

Fourteen Chinchilla (Chbb:CH) rabbits of either sex were randomisedeither to receive supplement with TTA fatty acids 800 mg daily (meanweight 3.6±0.09 kg) or to placebo (mean weight 3.5±0.47 kg).

The animals were pre-treated with TTA given as oral supplements, mixedwith daily food pellets, for three weeks to ensure accumulation of thefatty acid analogues in the tissues.

The local ethical committee for animal care and use approved theexperimental protocol.

TTA was synthesised as described in the method section. All otherchemicals were from common commercial sources and were of reagent grade.

Angioplasty Procedure

After premedication with 0,5 ml fentanyl (0.315 mg/ml) and fluanisone(10 mg/ml) (Hypnorm®) intramuscularly, the rabbits were anaesthetised byadministering diazepam 4 mg/kg intraperitoneally and maintained byadditional 0.3-0.4 ml of a 1:1 mixture of Hypnorm® and diazepam, usuallynecessary once during the procedure.

After local infiltration of the skin by lignocaine (Xylocaine®) andsurgical cut-down, the right carotid artery was cannulated by a 6Fsheath. A bolus of 100 U/kg of heparin was administered intraarterially.Before the angioplasty, an angiography was performed in the frontal viewby injecting 3 ml of ioxaglate (Hexabrix®) as contrast medium via aBerman® catheter.

An angioplasty balloon catheter (Express®) of 2.5 mm was positioned inthe proximal part of each iliac artery and balloon angioplasty wasperformed with 2 balloon inflations at the same site in each artery, at8 and 12 atmosphere for 30 seconds each to overstrech the artery. Theballoon marker, on the middle of the 20 mm long balloon, was placed overthe ilio-sacral joint, thus ensuring that the balloon was positioned atidentical sites. Inflation was performed using a pressure manometer(Encore® 26 inflation device, Scimed, Boston Scientific corporation)

A further angiography was performed in the same frontal view as thefirst angiography to assure artery patency. The carotid artery wasligated and the skin was closed with ligatures. Buprenorfin 0.3 mg(Temgesic®) and penicillin were given subcutaneous once daily for thefirst days.

A follow-up angiography was performed in the same frontal view after 10weeks, following the same procedure as indicated above, by usingioxaglate (Hexabrix®) as contrast medium. The sheath was now inserted inthe left carotid artery. Intravascular ultrasound was performed toquantitate wall thickness and lumen diameter. After angiography alaparotomy was performed and the abdominal aorta was cannulated with an18 G infusion-needle. The animals were euthanised by giving overdosepentobarbital intraarterially via the sheath in the left carotid artery.The iliac arteries were perfusion fixed by infusing 2% glutaraldehydeinto the distal aorta at a pressure of 100 mmHg over 15 minutes, using acannula in the inferior caval vein as efflux.

Quantitative Angiography

The frames with maximal opacification from baseline angiographies(before and after dilatation), as well as from the follow-up angiographywere stored for subsequent quantitative analysis. Arterial diameterbefore and after dilatation as well as the balloon to artery ratio weremeasured. The minimal luminal diameter, reference diameter and stenosiswere determined. All measurements were performed with a digitalelectronic calliper (Sandhill, model EC-1)(12). A balloon angioplastycatheter (Express® 2.5 mm), placed on the abdomen and dilated at 8atmospheres (rated balloon diameter 2.5 mm) served for calibration. Theresults are given in table 2.

TABLE 1 Angiographic measurements control before after angio- dilatationdilatation graphy placebo TTA placebo TTA placebo TTA diameter 1.85 ±0.20 1.91 ± 0.18 2.36 ± 0.19 2.36 ± 0.19 1.08 ± 0.47 1.60 ± 0.26 artery(mm) (D_(s)) reference 1.85 ± 0.20 1.91 ± 0.18 1.86 ± 0.30 1.80 ± 0.171.96 ± 0.21 2.07 ± 0.15 diameter (mm) (D_(r)) dilatation/ −27.7 −31.243.2 23.5 stenosis (D_(r) − D_(s))/D_(r) acute gain 0.51 ± 0.30 0.48 ±0.22 (mm) late loss 1.28 ± 0.49 0.76 ± 0.38 loss index 3.22 ± 2.27 1.53± 0.46

Acute gain: minimal luminal diameter (MLD) post dilatation minusdiameter artery before dilatation. Late loss: MLD post dilatation minusMLD at control angiography. Loss index: late loss divided by acute gain.

The results given in the table clearly demonstrate that the diameter ofthe arteries after the dilatation procedure are equal for the controland treated groups. However, at follow-up at six weeks the resultsindicate that the TTA has inhibited the decrease in artery diameter, andTTA is thus capable to prevent or reduce the formation of restenosis.

EXAMPLE 4

Prevention of Restenosis in Mini Pigs—Local Administration of TTA

Various local drug delivery systems have been developed to enable localapplication of pharmacological agents in conjunction with PTCA. In thisstudy 20 minipigs were randomised to placebo or active treatment withthe fatty acid analogue tetradecylthioacetic acid (TTA) via a Transport®multiphorous angioplasty balloon catheter. This catheter has a sleeveembracing the balloon and the drug is delivered through a separate lumenconnected to the sleeve which has multiple holes.

Coronary balloon angioplasty injury using an oversized balloon wasperformed to the LAD or LCx followed by 3 bolus deliveries of 0.5 ml(0.33 mg/ml TTA) of active drug/placebo. Quantitative angiography asindicated in the preceding sections was performed before and afterinjury, and after 4 weeks follow-up. Subsequently the pigs weresacrificed, perfusion-fixed with glutaraldehyde and the vessels preparedfor histology with computer assisted planimetry. Radiolabelled 1-¹⁴C-TTAwas administered locally in two extra pigs and confirmed the presence ofactive drug in the coronary arteries after 4 and 6 weeks.

The luminal diameter (mm) before, after and at follow-up (six weeksafter balloon injury) is indicated in table 2.

TABLE 2 Luminal diameter (mm) before and after PTCA, and at follow-upbefore after at follow-up placebo 2.7 3.0 1.3 TTA treated 2.6 3.2 2.2

The maximal intimal thickness was unchanged in the treated group withrespect to the placebo group.

The results are also visualised in the appending FIGS. 1 and 2 whichshows the Iliac artery 6 weeks after balloon injury for a control animaland a TTA administered animal, respectively.

TABLE 3 IVUS (intravascular ultrasound) Placebo TTA Area site mm² 0.42 ±0.086 0.61 ± 0.152 p = 0.008 Max. diameter 2.38 ± 0.311 2.87 ± 0.408 p =0.012 site mm Minimal dia- 2.25 ± 0.256 2.76 ± 0.381 p = 0.005 metersite mm

The ICUS (intracoronary ultrasound) results were consistent with theangiography data.

Histology

The dilated segments of the iliac arteries were located by fluoroscopy,using the ilio-sacral joint as anatomical hallmark and dissected inblock. Serial sections were processed and the segments were embedded inparafin. Cross sections were stained with hematoxylin-eosin andVerhoeff-van Gieson stains. All sections were evaluated for intimaproliferation, interruption of the internal elastic lamina, the presenceof luminal and intramural thrombus and for vessel area (data not shown).

EXAMPLE 5

Reduced Proliferation of Smooth Muscle Cells

Restenosis lesions may have an overgrowth of smooth muscle cells in theintiminal layer of the vessel, and we thus tested the effect of TTA oncultured muscle cells.

SMC are human smooth muscle cells available from American Type CultureCollection (Type 1999-CRL). The SMC cells were cultured in bottles (75cm²) in Ham's F12K medium. 24 hours after inoculation, the fatty acidsand BSA in a molar ratio of 1:2.5 were added to the medium, i.e. to afinal concentration of palmitic acid and TTA of 100 μM. After 3 and 6days the cells were trypsinated, and the number of cells determined byusing a light microscopy with counting chambers (Leitz Wetzler).

FIG. 3 shows the inhibiting effect of TTA on smooth muscle cells.

The inhibition of the proliferation of smooth muscle cells may be one ofseveral mechanisms for the restenosis preventing effect of TTA.

EXAMPLE 6

TTA Reduces the Level of Plasma Homocysteine

Increased levels of homocysteine, i.e. hyperhomocysteinemia has beenproposed to be associated with arterial diseases, and we thus measuredthe levels of homocysteine in plasma samples from Male Wistar rats given300 mg TTA per kg body weight per day in 10 days.

Total plasma homocysteine was measured by a fully automated fluorescenceassay. 30 μl plasma was reduced by 30 μl NaBH4/DMSO solution (6 mol/L).After 1,5 min 20 μl of the fluorescence reagent monobromobimane (25mmol/L) in acetonitrile was added and allowed to react for 3 min. 20 μlof the sample was then immediately analysed with HPLC by injection on astrong cation-exchange column, and then by column switching into acyclohexyl silica column. The SCX column was eluted isocratically andthe CH column was eluted with a linear methanol gradient (17-35% in 5min) in 20 mmol/L formate buffer. The homocysteine was eluted at aretention time of 4.5 min. The results are given in table 4.

TABLE 4 Plasma concentration of homocysteine Plasma concentration(μmol/L) Control (CMC) 10.6 ± 0.8  TTA 5.4 ± 1.0

What is claimed is:
 1. A method for a therapeutic treatment andprophylaxes of restenosis in a mammal, said method comprisingadministering to a mammal to susceptible to a formation of restentherapeutically effective amount of at least one fatty acid analogue ofthe general formula (I) CH₃—[CH₂]_(m)—[X_(i)—CH₂]_(n)—COOR wherein n isan integer from 1 to 12, and m is an integer from 0 to 23, and i is anodd number which indicates the position relative to COOR, and each X_(i)is independently selected from the group consisting of O, S, SO, SO₂,Se, and CH₂, and R represents hydrogen or C₁-C₄ alkyl, with the provisothat at least one of the X_(i) is not CH₂, or a salt, prodrug, orderivative thereof.
 2. The method of claim 1, wherein the mammal is ahuman.
 3. The method of claim 1, wherein m is greater than or equal to13.
 4. The method of claim 2, wherein X_(i=3) is selected from the groupconsisting of O, S, SO, SO₂, and Se, and wherein X_(i=5-25) is CH₂. 5.The method of claim 4, wherein X_(i=3) is S.
 6. The method of claim 4,wherein X_(i=3) is Se.
 7. The method of claim 1, wherein the at leastone fatty acid analogue is administered such that its concentration ismaintained substantially continuously in the blood of the mammal for theduration of the period of administration.
 8. The method of claim 1,wherein the at least one fatty acid analogue is administered orally orlocally.
 9. The method of claim 1, wherein the restenosis is due to agraft or transplant procedure.
 10. The method of claim 1, wherein therestenosis is due to angioplasty.
 11. The method of claim 10, whereinthe angioplasty is percutaneous transluminal coronary angioplasty(PTCA).
 12. A method for lowering a concentration of homocysteine inplasma, comprising administering to a mammal an effective amount of atleast one fatty acid analogue of the general formula (I)CH₃—[CH₂]_(m)—[X_(i)—CH₂]_(n)—COOR wherein n is an integer from 1 to 12,and m is an integer from 0 to 23, and i is an odd number which indicatesthe position relative to COOR, and each X_(i) is independently selectedfrom the group consisting of O, S, SO, SO₂, Se, and CH₂, and Rrepresents hydrogen or C₁-C₄ alkyl, with the proviso that at least oneof the X_(i) is not CH₂, or a salt, prodrug, or derivative thereof. 13.The method of claim 12, wherein the mammal is a human.
 14. The method ofclaim 12, wherein m is greater than or equal to
 13. 15. The method ofclaim 13, wherein X_(i=3) is selected from the group consisting of O, S,SO, SO₂, and Se, and wherein X_(i=5-25) is CH₂.
 16. The method of claim15, wherein X_(i=3) is S.
 17. The method of claim 15, wherein X_(i=3) isSe.
 18. The method of claim 12, wherein the at least one fatty acidanalogue is administered such that its concentration is maintainedsubstantially continuously in the blood of the mammal for the durationof the period of administration.
 19. The method of claim 12, wherein theat least one fatty acid analogue is administered orally or locally.